禽偏肺病毒C型核衣壳N蛋白的哺乳动物细胞表达系统表达及鉴定

利用哺乳动物细胞表达系统对禽偏肺病毒C型核衣壳N蛋白进行表达,并检测所表达融合蛋白的抗原性。首先将N基因克隆到真核表达载体pEGFP-C1中,构建重组真核表达载体,然后将阳性重组质粒转染BHK-21细胞,通过倒置荧光显微镜和共聚焦显微镜检测N蛋白的表达,并用Western blot检测表达蛋白的抗原性。结果显示,禽偏肺病毒核衣壳N蛋白主要表达在细胞浆中,在转染后48h后表达量最高;与抗N蛋白抗体有抗原性,所表达核衣壳N融合蛋白大小为72kDa左右,与预期结果一致。结论,本试验成功应用哺乳动物表达系统表达了禽偏肺病毒C型核衣壳N蛋白,表达蛋白具有抗原性,为与禽偏肺病毒核衣壳N蛋白的相关研究奠定了基础。

Mammalian expression and identification of avian metapneu movirus subgroup C nucleocapsid protein

The aims of this paper were to express subgroup C avian metapneumovirus virus(aMPV)nucleocapsid(N)protein by Eukaryotic expression system and to further analyze the antigenicity of the recombinant protein.The N gene was cloned into eukaryotic expression pEGFP-C1 vector to obtain the recombinant expression vector.The positive recombinant expression vector was transfected into BHK-21 cells.The recombinant N protein expression was analyzed by confocal microscopy and inverted fluorescence microscope and the antigenicity of the recombinant protein by western-blotting.The results demonstrated that the N protein expressed mainly in the cytoplasm and exhibited a specific and single band with a size of about 72 kDa when reacted with a monoclonal antibody raised against subgroup C aMPV N protein.In conclusion,this study expressed aMPV N protein in mammalian expression system with a good antigenicity,which made the foundation work for the relatied study of aMPV N protein.